Development of Combinatorial Therapeutics for Spinal Cord Injury using Stem Cell Delivery

Résumé

In this study, nerve-mimetic composite hydrogels were developed and characterized that can be utilized to investigate and capitalize on the pro-regenerative behavior of adipose-derived stem cells for spinal cord injury repair.

Résumé

Traumatic spinal cord injury (SCI) induces permanent sensorimotor deficit below the site of injury. It affects approximately a quarter million people in the US, and it represents an immeasurable public health concern. Research has been conducted to provide effective therapy; however, SCI is still considered incurable due to the complex nature of the injury site. A variety of strategies, including drug delivery, cell transplantation, and injectable biomaterials, are investigated, but one strategy alone limits their efficacy for regeneration. As such, combinatorial therapies have recently gained attention that can target multifaceted features of the injury. It has been shown that extracellular matrices (ECM) may increase the efficacy of cell transplantation for SCI. To this end, 3D hydrogels consisting of decellularized spinal cords (dSCs) and sciatic nerves (dSNs) were developed at different ratios and characterized. Histological analysis of dSCs and dSNs confirmed the removal of cellular and nuclear components, and native tissue architectures were retained after decellularization. Afterward, composite hydrogels were created at different volumetric ratios and subjected to analyses of turbidity gelation kinetics, mechanical properties, and embedded human adipose-derived stem cell (hASC) viability. No significant differences in mechanical properties were found among the different ratios of hydrogels and decellularized spinal cord matrices. Human ASCs embedded in the gels remained viable throughout the 14-day culture. This study provides a means of generating tissue-engineered combinatorial hydrogels that present nerve-specific ECM and pro-regenerative mesenchymal stem cells. This platform can provide new insights into neuro-regenerative strategies after SCI with future investigations.

Introduction

Approximately 296,000 people are suffering from traumatic SCI, and every year there are about 18,000 new SCI cases occurring in the U.S.A.¹. Traumatic SCI is commonly caused by falls, gunshot wounds, vehicle accidents, and sports activities and often causes permanent loss of sensorimotor function below the site of injury. The estimated lifetime expenses for SCI treatment range between one to five million dollars per individual with significantly lower life expectancies¹. Yet, SCI is still poorly understood and largely incurable, mainly due to complex pathophysiological consequences after the injury². Various strategies have been investigated, including cell transplantation and biomaterials-based scaffolds. While transplantation of cells and biomaterials has demonstrated potential, the multifaceted nature of SCI suggests that combinatorial approaches may be more beneficial³. As a result, many combinatorial strategies have been investigated and demonstrated better therapeutic efficacy than individual components. However, further studies are needed to provide novel biomaterials for delivering cells and drugs³.

One promising approach to fabricating natural hydrogels is tissue decellularization. The process of decellularization utilizes ionic, non-ionic, physical, and combinatorial methods to remove all or most cellular and nucleic materials while preserving ECM components. By removing all or most of the cellular components, ECM-derived hydrogels are less immunoreactive to the host after implantation/injection⁴. There are several parameters to measure in order to assess the quality of decellularized tissues: removal of cellular/nucleic contents, mechanical properties, and ECM preservation. The following criteria have been established to avoid adverse immune responses: 1) less than 50 ng double-stranded DNA (dsDNA) per mg ECM dry weight, 2) less than 200 bp DNA fragment length, and 3) almost or no visible nuclear material stained with 4'6-diamidino-2-phenuylindole (DAPI)⁵. Mechanical properties can be quantified by tensile, compression, and/or rheology tests, and they should be similar to the original tissue⁶. In addition, protein preservation can be evaluated by proteomics or quantitative assays focusing on the main components of decellularized tissues, for instance, laminin, glycosaminoglycan (GAG), and chondroitin sulfate proteoglycan (CSPG) for the spinal cord^7,8. Verified ECM-derived hydrogels can be recellularized with different types of cells to aid cell-based therapy⁹.

A variety of cell types, such as Schwann cells, olfactory ensheathing cells, bone-marrow-derived mesenchymal stem cells (MSCs), and neural stem/progenitor cells, have been studied for SCI repair^10,11,12. However, clinical use of these cells is limited due to ethical concerns, sparse integration with neighboring cells/tissues, lack of tissue sources for high yield, inability to self-renew, and/or limited proliferative capacity^13,14,15. Unlike these cell types, human adipose-derived MSCs (hASCs) are an attractive candidate because they are easily isolated in a minimally invasive manner using lipoaspirates, and a large number of cells can be obtained¹⁶. In addition, hASCs have the ability to secrete growth factors and cytokines that have the potential of neuroprotective, angiogenetic, wound healing, tissue regeneration, and immunosuppression^{17,18,19,20,21}.

As was described, multiple studies have been conducted^22,23,24, and a lot has been learned from them, but heterogeneous characteristics of SCI have limited their efficacy in promoting functional recovery. As such, combinatorial approaches have been proposed to increase treatment efficacy for SCI. In this study, composite hydrogels were developed by combining decellularized spinal cords and sciatic nerves for a three-dimensional (3D) hASC culture. Successful decellularization was confirmed by histological and DNA analyses, and different ratios of nerve composite hydrogels were characterized by gelation kinetics and compression tests. The viability of hASCs in the nerve composite hydrogels was investigated to prove that this hydrogel can be utilized as a 3D cell culture platform.

Protocole

The porcine tissues were commercially obtained, so approval was not required by the animal ethics committee.

1. Decellularization of porcine spinal cords (Estimated time: 5 days)

NOTE: Perform the decellularization using previously established protocols with modifications^25,26. All procedures should be done in a sterile biosafety cabinet at room temperature unless stated otherwise. All solutions should be sterile filtered using a bottle top filter (0.2 µm pore size) into autoclaved bottles. Procedures to be carried out at 37 °C can be done inside an incubator or a clean oven set to 37 °C.

1. Preparation of decellularization solutions
   NOTE: All solutions are calculated for 1 L. Users may need to adjust the final required volume according to their experimental needs.
   1. Dilute 500 mL of 0.05% trypsin/ ethylenediaminetetraacetic acid (EDTA) with 500 mL of phosphate-buffered saline (PBS) to make 0.025% trypsin/EDTA.
   2. Dilute 300 mL of 10% Triton X-100 with 700 mL of PBS to make 3% Triton X-100. Mix 0.56 g of NaCl, 1.31 g of NaH₂PO₄H₂O, and 10.85 g of HNa₂O₄P·7H₂O in 1 L of deionized water to make 100 mM Na/50 mM phos buffer.
   3. Mix 32.4 g of sucrose in 1 L of deionized water to make 1 M sucrose. Mix 40 g of sodium deoxycholate (SD) in 1 L of deionized water to make 4% SD solution. Dilute 6.7 mL of 15% peracetic acid with 993.3 mL of 4% ethanol.
2. Preparation of porcine spinal cord
   NOTE: Spinal cords were shipped frozen without any solution and kept at -80 °C until use.
   1. Thaw spinal cord at 4 °C in the fridge for 18-24 h before decellularization. Use sterile scissors to remove dura mater carefully.
   2. Cut the spinal cord into small pieces (approximately 1 cm long). Place one piece into a 15 mL tube or a maximum of three pieces into a 50 mL tube.
3. Decellularization of spinal cord
   NOTE: After each step, decellularization solutions are manually poured into a large beaker to be discarded. Small autoclavable stainless-steel strainer can be used to help discard decellularization solutions without losing the tissues needed for each step. Spinal cord was agitated at 83 rpm unless stated otherwise.
   1. Rinse the spinal cord with deionized water for 18-24 h at 4 °C and 60 rpm.
   2. Rinse the spinal cord with 0.025% trypsin/EDTA for 1 h at 37 °C and 40 rpm. Then, rinse the spinal cord with PBS for 15 min, 2x.
   3. Rinse the spinal cord with 3% Triton X-100 for 2 h. Rinse the spinal cord with 100 mM Na/50 mM phos buffer for 15 min, 2x.
   4. Rinse the spinal cord with 1 M sucrose for 1 h. Then, rinse the spinal cord with deionized water for 1 h.
   5. Rinse the spinal cord with 4% SD for 2 h. Then, rinse the spinal cord with 100 mM Na/50 mM phos buffer for 15 min, 2x.
   6. Rinse the spinal cord with 0.1% peracetic acid in 4% ethanol for 4 h. Then, rinse the spinal cord with PBS for 1 h.
   7. Rinse the spinal cord with deionized water for 1 h, 2x. Then, rinse the spinal cord with PBS for 1 h.
   8. Lyophilize the spinal cord at 0.01 mbar and -56 °C for 3 days and store dry until use.

2. Decellularization of porcine sciatic nerve (Estimated time: 5 days)

NOTE: Perform the decellularization using a previously established protocol²⁷. All procedures should be done in a sterile biosafety cabinet at room temperature unless stated otherwise. All solutions should be sterile filtered using a bottle top filter (0.2 µm pore size) into autoclaved bottles. Procedures to be carried out at 37 °C can be done inside an incubator or a clean oven set to 37°C.

1. Preparation of decellularization solutions
   NOTE: All solutions are calculated for 1 L. Users may need to adjust the final required volume according to their experimental needs.
   1. Prepare 50 mM Na/10 mM phos buffer by mixing 1.86 g of NaCl, 0.262 g of d NaH₂PO₄H₂O, and 2.17 g of HNa₂O₄P·7H₂O in 1 L of deionized water.
   2. Prepare 125 mM sulfobetaine-10 (SB-10) solution by mixing 38.4 g of SB-10 in 1 L of 50 mM Na/10 mM phos buffer.
   3. Prepare 3 % SD/0.6 mM sulfobetaine-16 (SB-16) solution by mixing 30 g of SD and 0.24 g of SB-16 in 1 L of 50 mM Na/10 mM phos buffer.
2. Preparation of porcine sciatic nerve
   NOTE: Sciatic nerves were shipped frozen with PBS and kept at -80 °C until use.
   1. Thaw the sciatic nerve at 4 °C in the fridge 18-24 h before decellularization.
   2. Cut sciatic nerve into small pieces (approximately 1 cm long). Place one piece into a 15 mL tube or a maximum of three pieces into a 50 mL tube.
3. Decellularization of the sciatic nerve
   NOTE: After each step, decellularization solutions are manually poured into a large beaker to be discarded, and a small autoclavable stainless-steel strainer can be used to help discard decellularization solutions without losing the tissues needed for each step. The sciatic nerve was agitated at 15 rpm unless stated otherwise.
   1. Rinse the sciatic nerve with deionized water for 7 h. Then, rinse the sciatic nerve with 125 mM sulfobetaine-10 (SB-10) in 50 mM Na/10 mM phos buffer for 18 h.
   2. Rinse the sciatic nerve with 100 mM Na/50 mM phos buffer for 15 min. Then, rinse the sciatic nerve with 3% SD/0.6 mM sulfobetaine-16 (SB-16) in 50 mM Na/10 mM phos buffer for 2 h.
   3. Rinse the sciatic nerve with 100 mM Na/50 mM phos buffer for 15 min, 3x. Then, rinse the sciatic nerve with 125 mM SB-10 in 50 mM Na/10 mM phos buffer for 7 h.
   4. Rinse the sciatic nerve with 100 mM Na/50 mM phos buffer for 15 min. Then, rinse the sciatic nerve with 3% SD/0.6 mM SB-16 in 50 mM Na/10 mM phos buffer for 1.5 h.
   5. Rinse the sciatic nerve with 50 mM Na/10 mM phos buffer for 15 min, 3x. Then, rinse the sciatic nerve with 75 U/mL of deoxyribonuclease (DNase) for 3 h without agitation.
   6. Rinse the sciatic nerve with 50 mM Na/10 mM phos buffer for 1 h, 3x. Then, rinse the sciatic nerve with 0.2 U/mL of Chondroitinase ABC for 16 h without agitation at 37 °C.
   7. Rinse the sciatic nerve with PBS for 3 h, 3x. Lyophilize the sciatic nerve at 0.01 mbar and -56 °C for 3 days and store dry until use.

3. Digestion of decellularized tissues and fabrication of composite hydrogels (Estimated time: 4 days)

1. Chop or grind the decellularized tissues into powder by using scissors or homogenizer. Sterilize the tools to chop or grind the tissues using an autoclave at 121 °C for 45 min. The ethylene oxide method is also applicable.
2. Digest the tissues separately in 0.01 N hydrochloric acid (HCl) solution containing 1 mg/mL pepsin at a concentration of 15 mg/mL. The estimated weights of decellularized spinal cord and sciatic nerve are 50-100 mg and 100-150 mg per piece, respectively.
3. Place the magnetic bar and stir at 500 rpm and 4 °C for at least 4 days to generate pregel solutions.
4. Mix sciatic nerve and spinal cord pregel at following volumetric ratios: 2:1, 1:1, and 1:2. Adjust pH 7.4 using 1 N sodium hydroxide (NaOH) and HCl and dilute to the desired concentration using M199 media and 1x PBS. Incubate at 37 °C for 30 min.
   NOTE: Add NaOH 1 µL at a time. M199 media can be used as a pH indicator since it turns light pink when the pH is 7.4, but pH strips should be used to confirm the pH level.

4. Verification of decellularization

1. Hematoxylin and Eosin staining (H&E; Estimated time: 8 days)
   NOTE: After each rinse step, the solutions are manually poured into a large beaker to be discarded.
   1. Fix fresh and decellularized spinal cord and sciatic nerve in 3.7% formaldehyde at 4 °C for 18 h. Place one piece in a 15 mL tube.
   2. Remove formaldehyde and place the tissues in 10% sucrose for 1 day. Remove 10% sucrose and place the tissues in 30% sucrose for 6 days.
   3. Fill an appropriately sized cryomold with optimal cutting temperature (OCT) halfway. Place the decellularized tissues, cover them with OCT, and allow them to absorb the OCT for 1 day.
   4. After 1 day, freeze the tissues overnight at -80 °C. Cryosection the tissues at 10 µm thickness using a cryostat.
   5. Rinse with 1x PBS for 5 min, 2x. Then, rinse with tap water for 5 min.
   6. Stain with hematoxylin solution for 1 min. Rinse with tap water for 1 min, 3x.
   7. Stain with eosin solution for 1 min. Rinse with 95 % ethanol for 1 min, 2x and with 100% ethanol for 1 min, 3x.
   8. Rinse with xylene for 1 min, 2 min, and then 1 min. Let the slides dry for 5 min.
   9. Use a cotton swab to drip 3 - 4 drops of dibutylphthalate polystyrene xylene (DPX) mount solution onto the slides. Place the coverslips on top of DPX-covered slides. Let the slides dry overnight.
2. DNA analysis (Estimated time: 1 h)
   1. Weigh decellularized and lyophilized tissues. Isolate and quantify DNA using commercially available kits according to the manufacturer's instructions.

5. Characterization of composite hydrogels

1. Gelation turbidity test (Estimated time: 1 h)
   1. Place 100 µL of pregel solutions in each well of a 96-well plate on ice. Read absorbance at 405 nm every 2 min for 45 min using a plate reader.
   2. Calculate normalized absorbance using the following equation:
      (Absorbance - Absorbance_Initial) / (Absorbance_Maximum - Absorbance_Initial)
   3. Calculate the slope of the curve and the time to achieve 50% and 95% gelation, t_1/2 and t₉₅, respectively.
2. Compression test (Estimated time: 1 min per hydrogel)
   1. Fabricate composite hydrogels at the concentration of 12 mg/mL with 8 mm diameter and 2 mm height.
   2. Use a rheometer to compress the samples with a load of 250 N in between stainless-steel parallel plates at a strain rate of 10% of sample height/min. The software that provides rheometer readings will provide a stress-strain curve by applying the force and measuring the strain until the hydrogels break.
   3. Calculate Young's modulus from the slope of the linear region of the stress-strain curves.

6. Three-dimensional culture of human ASCs in nerve composite hydrogels

1. Preparation of three-dimensional (3D) platform (Estimated time: 3 h)
   1. Etch silicon wafer with SU-8 photoresist to generate circular patterns of 200 µm in depth and 4 mm in diameter.
   2. Mix polydimethylsiloxane (PDMS) base and curing agent at a ratio of 10:1. Pour the mixture onto the silicon wafer and let it sit for 20 min to remove all the bubbles that arise from mixing the PDMS base and curing agent.
   3. Place it in the oven and cure for 2 h at 70 °C. Demold the cured PDMS sheet and punch out using an 8 mm diameter biopsy punch to make PDMS microwells.
   4. Place microwells in a 96-well plate and put the well plate in an air-plasma cleaner to sterilize PDMS microwells.
   5. Functionalize PDMS microwells with 1% polyethyleneimine (PEI) and 0.1% glutaraldehyde (GA) for 10 min and 20 min, respectively. Wash microwells with distilled water, 2x.
2. Preparation of hASCs (Estimated time: 7 days)
   1. Culture passage cells for 2-5 passages in hASCs growth media (hASCs basal media supplemented with fetal bovine serum (FBS) and penicillin/streptomycin (Pen-strep)) until confluent. Passage and calculate the number of cells by using a hemacytometer or cell counter.
3. ASC-laden nerve composite hydrogels (Estimated time: 2 h)
   1. Mix sciatic nerve and spinal cord pregel at a volumetric ratio of 2:1, 1:1, 1:2, and prepare spinal cord-only hydrogel without mixing in any sciatic nerve pregel.
   2. Add M199 media and adjust pH 7.4 using 1 N NaOH and HCl. Resuspend hASCs with growth media in the pregel at a density of 1 x 10⁶ cells/mL.
      NOTE: The amount of M199 and cell suspension should be 10% of pregel.
   3. Dilute the pregel to 12 mg/mL using 1x PBS. Place the pregel onto microwells and incubate for 30 min. Culture ASC-laden hydrogels in hASCs growth media.
4. Cell viability test (Estimated time: 4 h per day)
   1. Prepare viability test solutions using commercially available reagents according to the manufacturer's instructions.
   2. Aspirate media on days 1, 4, 7, and 14. Add reagent to the wells and incubate at 37 °C for 3 h following the manufacturer's instructions.
   3. Read fluorescence at excitation/emission of 560/590 nm using a plate reader. Calculate the percentage difference using the following equation:
      [(fluorescence_sample- fluorescence_blank) / fluorescence_blank] x 100

Résultats

Decellularized tissues were prepared using previously established protocols with slight modifications^26,27. After decellularization, lyophilization, and digestion, nerve composite hydrogels at ratios of SN:SC = 2:1, 1:1, 1:2, and spinal cord-only hydrogels were fabricated (Figure 1). Removal of nuclear components was confirmed by H&E staining (Figure 2A). To quantitatively assess the decellularizatio...

Discussion

It is widely believed that the pathophysiology of SCI is complex and multifaceted. Even though single therapies such as cell transplantation, drug delivery, and biomaterials each have provided valuable insights into SCI, the complicated nature of SCI may limit their individual efficacy^28,29,30,31. Therefore, efforts to develop effective combinatorial therapeutics have increased. The nerve compo...

matériels

Name	Company	Catalog Number	Comments
3-(Decyldimethylammonio)propane sulfonate inner salt	Sigma-Aldrich	D4266	Used during sciatic nerve decellularization, SB-10
3-(N,N-Dimethylpalmitylammonio)propane sulfonate	Sigma-Aldrich	H6883	Used during sciatic nerve decellularization, SB-16
AlamarBlue reagent	Fisher Scientific	DAL1100	Used during AlamaBlue cell viabiiltiy test
Chondroitinase ABC	Sigma-Aldrich	C3667	Used during sciatic nerve decellularization
Cryostat	Leica	CM1860
Deoxyribonuclase	Sigma-Aldrich	D4263	Used during sciatic nerve decellularization
Disodium hydrogen phosphate heptahydrate	VWR	BDH9296	Chemical for 100 mM Na/50 mM phos and 50 mM Na/10 mM phos buffer
DNeasy Blood & Tissue kit	Qiagen	69506	Used during DNA analysis
Dpx Mountant for histology,slide mounting medium	Sigma-Aldrich	6522	Used during H&E staining
Eosin	Sigma-Aldrich	HT110216	Used during H&E staining
Ethanol	VWR	89125-172
Formaldehyde	Sigma-Aldrich	252549	Used during H&E staining
Glutaraldehyde (GA)	Sigma-Aldrich	G6257	Used during PDMS surface functionalization
hASC growth media	Lonza	PT-4505	Used to culture hASCs, containing of fetal bovine serum and penicilin/streptomycin
Hematoxylin	VWR	26041-06	Used during H&E staining
human adipose-derived stem cell	Lonza	PT-5006
Hydrochloric acid (HCl)	Sigma-Aldrich	320331	Used to digest decellularizied tissues and adjust pregels solutions
M199 media	Sigma-Aldrich	M0650	Used to dilute pregels to desired concentration
Optimal cutting temperatue compound	Tissue-Tek	4583
Pepsin	Sigma-Aldrich	P7000	Used to digest decellularized tissues
Peracetic acid	Lab Alley	PAA1001	Used during spinal cord decellularization
Phosphate buffered saline (PBS)	VWR	97062-948
Plate reader	BioTek Instruments	Synergy Mx
Polyethyleneimine (PEI)	Sigma-Aldrich	181978	Used during PDMS surface functionalization
Porcine sciatic nerve	Tissue Source LLC		Live pigs, with no identifiable information and no traceability details
Porcine spinal cord	Tissue Source LLC		Live pigs, with no identifiable information and no traceability details
QuantiFluor dsDNA system	Promega	E2670	Used to analyze DNA contents
Rheometer	TA Instruments	DHR 2
Rugged rotator	Glas-co	099A RD4512	Used during spinal cord decellularization
Sodium chloride (NaCl)	VWR	BDH9286	Chemical for 100 mM Na/50 mM phos and 50 mM Na/10 mM phos buffer
sodium deoxycholate	Sigma-Aldrich	D6750
Sodium dihydrogen phosphate monohydrate	VWR	BDH9298	Chemical for 100 mM Na/50 mM phos and 50 mM Na/10 mM phos buffer
Sodium hydroxide solution (NaOH)	Sigma-Aldrich	415443	Used to adjust pregels solutions
SU-8	Kayaku advnaced materials	SU8-100	Used to coat silicon wafer
Sucrose	Sigma-Aldrich	S8501	Used during spinal cord decellularization
Sylgard 184 silicone elastomer kit	DOW	1317318	Polydimethylxiloxane (PDMS) base and curing agent
Triton X-100	Sigma-Aldrich	X100	Used during spinal cord decellularization
Trypsin-EDTA (0.05%), phenol red	Thermo Fisher	25300062	Used during hASC work and during spinal cord decellularization
Tube revolver rotator	Thermo Fisher	88881001	Used during sciatic nerve decellularization
Xylene	VWR	MK866816	Used during H&E staining